Changes in molecular structure of biodegradable plastics during degradation in soils estimated by FT-IR and NMR

Among the biodegradable plastic specimens (poly-(3-hydroxy-butylate-valerate) (PHB / V), poly-(ε-caprolactone) (PCL), poly-(butylene succinate) (PBS), poly-(butylene succinate and adipate) (PBSA), and poly-lactide (PLA)) that were placed in soils for 1 year at nineteen sites in Japan, plastic specimens with appreciable biodegradation were studied for the transformation of the chemical structure by FT-IR, ¹H-NMR, and ¹³C-NMR. No appreciable differences in the main absorbency-bands of the atomic groups were recognized by FT-IR for any of the plastic specimens tested. However, both ¹H-NMR and ¹³C-NMR analyses suggested that molecular structure of the PHB / V specimens changed after 1 year placement in soils. Based on the assignment of the respective signals of chemical shifts derived from valerate, selective degradation of the valerate moiety in the PHB / V specimens was observed. In contrast, although weight loss, and/or a decrease in tensile strength and elongation were observed after the placement in soils for the PCL, PBS, PBSA, and PLA specimens, the analyses of these specimens by FT-IR, ¹H-NMR, and ¹³C-NMR did not reveal any changes in their molecular structure.     

Production of bioactive bovine fibroblast growth factor 4 in E. coli based on the common nucleotide sequence of its structural gene in three breeds

Fibroblast growth factor 4 (FGF4) is considered a crucial gene in the proper development of bovine embryos. We recently determined the FGF4 gene sequence in eight cattle derived from three breeds and revealed a common nucleotide sequence of the structural gene encoding FGF4, which leads to the deletion and mutation of amino acid sequences in the mature FGF4 (Pro³²‐Leu²⁰⁶) compared with the sequence previously reported. In the present study, HisbFGF4, a 6× histidine‐tagged bovine FGF4 (Pro³²‐Leu²⁰⁶), was produced in Escherichia coli based on the validated nucleotide sequence and purified by heparin column chromatography. In primary bovine fibroblasts, HisbFGF4 showed significant mitogenic activity, whereas, intriguingly, the activity of a commercially available recombinant human FGF4 (Gly²⁵‐Leu²⁰⁶) produced in E. coli was weaker than that of HisbFGF4. In conclusion, the present study provides a simple method for the production of a bioactive bovine FGF4 derivative in E. coli utilizing its structural gene elucidated by us.     

Effects of winter buds on winter nitrogen uptake and allocation in Pinus densiflora saplings

Studies of nitrogen (N) use by plants have confirmed some winter N uptake; however, the mode of regulation of plant N use in winter is unknown. The regulation of N use by plants during winter may differ from that in the growing season, as plant growth strongly affects N use. We investigated the effects of winter buds on winter N use by Japanese red pine (Pinus densiflora), as a previous study demonstrated that N absorbed during winter contributes significantly to leaf growth in the following spring. We conducted a bud pruning experiment during winter to examine the effects of winter buds on winter N uptake and allocation among plant organs using ¹⁵N labeling. Over a three-week labeling period, the ¹⁵N content in roots increased to 0.20 ± 0.12 mg N g DW^?1, which is equivalent to 1.8 ± 1.1 % of the total N content in the roots. However, this absorbed ¹⁵N rarely appeared in needles and buds. Bud pruning did not affect ¹⁵N uptake and allocation. On the other hand, significant total N retranslocation was found within the crowns of saplings without bud pruning, but N was not retranslocated in bud-pruned plants. The bud pruning experiment indicated that N was retranslocated from needles into winter buds. Since soil N availability changes dramatically and is unstable in many forest ecosystems, N contained in needles would be a more stable source of N than newly absorbed N.     

Algicidal effect of 3-(3-indolyl)butanoic acid, a control agent of the bacterial wilt pathogen, Ralstonia solanacearum

The chemical control agent 3-(3-indolyl)butanoic acid, previously reported as a control agent for the bacterial wilt pathogen Ralstonia solanacearum, was shown to suppress the growth of green algae during hydroponic culture of tomato. The algicidal activity of the compound was effective at 10 μg/ml, completely preventing generation of green algae under non-shaded greenhouse conditions. The algicidal effect was mainly due to suppression of the growth of motile unicellular algal cells tentatively identified as Chlamydomonas spp., which are commonly occurring in the hydroponic solution and vigorously multiply to form an algal mat on the sponge supports. The compound has potential as a non-phytotoxic algicide for hydroponically cultured crop plants.     

Hydroxytyrosol induces proliferation and cytoprotection against oxidative injury in vascular endothelial cells: role of Nrf2 activation and HO-1 induction

Hydroxytyrosol (HT), a phenolic compound in olive oil and leaves, has been reported to prevent various human pathologies including cardiovascular diseases. This study investigated the effects of HT on proliferation and protection against oxidative stress-induced damage in vascular endothelial cells (VECs) and the molecular mechanism(s) involved. Treatment of VECs with HT increased cell proliferation, promoted wound repair, and protected cells against H(2)O(2) cytotoxicity through the activation of Akt and ERK1/2, but not p38 MAPK. HT increased the expression and nuclear translocation of nuclear factor-E2-related factor-2 (Nrf2). Nrf2 expression was attenuated by LY294002 and U0126, inhibitors of phosphatidylinositol-3-kinase and MEK1/2, respectively. Nrf2 siRNA decreased both proliferative and cytoprotective effects of HT and abrogated HO-1 induction. Moreover, HO-1 inhibition with HO-1 siRNA or zinc protoporphyrin IX significantly prevented HT-induced cell proliferation, cytoprotection, and reduction in intracellular reactive oxygen species (ROS), suggesting that HO-1 is involved in these HT functions. The findings demonstrate that HT positively regulates the antioxidant defense system in VECs through the activation of Nrf2 followed by cell proliferation and resistance to vascular injury. The present study provides a molecular basis for the contribution of HT in the Mediterranean diet to the prevention of cardiovascular diseases.     

Detection of Culicoides brevitarsis activity in Kyushu

Culicoides brevitarsis transmits important ruminant arboviruses, such as Akabane, Aino and bluetongue viruses. The presence of this species has so far been recognized primarily in Okinawa, the southernmost prefecture of Japan. In entomological surveys in 2008 and 2009, C. brevitarsis was collected at 8 sites throughout Nagasaki, Kumamoto and Kagoshima Prefectures. The collection sites are all located near pastures, where the larvae of C. brevitarsis can grow in cattle dung left in the field. C. brevitarsis was confirmed at the same sites in two consecutive years, suggesting that it overwinters in Kyushu. Given the risk of arbovirus transmission, the ecology of C. brevitarsis in Japan, such as its distribution range, seasonal abundance and larval breeding sites, should be investigated in more detail.     

Distribution of glycoproteins on feline testicular sperm, epididymal sperm and ejaculated sperm

Glycoproteins (GPs) are known to be involved in the phenomenon of sperm maturation and capacitation. In the present study, we investigated the attachment of GPs on sperm cell membrane during the process of feline sperm maturation from testicular sperm to ejaculated sperm by using 8 FITC-labeled lectins. The results showed that 3 types of GPs were presented on testicular sperm and 7 on caput epididymal sperm. Corpus and cauda epididymal sperm and ejaculated sperm had GPs detected by 8 FITC-labeled lectins used in the present study. This study demonstrates the part of the characteristic of GPs that are present on the feline sperm cell membrane during the process of sperm maturation.     

Effects of downregulating DNA methyltransferase 1 transcript by RNA interference on DNA methylation status of the satellite I region and in vitro development of bovine somatic cell nuclear transfer embryos

For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency.